New diacylglycerol-lactones continued to be studied with the aim of targeting individual protein kinase C isozymes and other diacylglycerol-responsive proteins containing similar C1 domains. The two DAG-lactones selected for consideration by the JDC of the Molecular Targets Faculty were synthesized in larger scale and tested against the NCI 60-cell line panel. The compounds are still strong candidates for further consideration pending results from additional studies by Dr. Kevin A. Camphausen, Head, Imaging and Molecular Therapeutics Section, NCI. Additional diacylglycerol lactones built with a rigid 4-[(methylphenyl)ethynyl]phenyl rod separated from the exocyclic acylcarbonyl of the DAG-lactone core by a spacer unit of variable length were synthesized and studied. Binding affinities for a panel of classical and novel PKC isozymes in two different phospholipids environments, one corresponding to the plasma membrane of cells, were determined. The kinetics and translocation sites for the PKC isozymes &amp;#945;and &amp;#948;upon treatment with the compounds were studied as well as the early response of ERK phosphorylation and the late response of induction of apoptosis in the human prostatic carcinoma cell line LNCaP. It was found that the different spatial disposition of the rigid structural motif on the DAG-lactones contributed to differential activity.